This section provides background information related to the present disclosure which is not necessarily prior art.
A current limitation in biology, biomedical engineering, and related fields is an inability during seeding on a growth surface to segregate populations of varying cell types, as well as selectively placing cells only in specific areas, without the use of chemical or permanent physical surface modifications.
Using arrays of micropatterned wells, groups have been able to separate individual cells into physical compartments (see, e.g., Jacqueline R. Rettig and Albert Folch, Large-Scale Single-Cell Trapping And Imaging Using Microwell Arrays, Analytical Chemistry, 77 (17), 2005, 5628-5634) or several cells into physical compartments (see, e.g., Jeffrey C. Mohr, Juan J. de Pablo, Sean P. Palecek, 3-D Microwell Culture of Human Embryonic Stem Cells, Biomaterials, 27 (36), 2006, 6032-6042). These methods are beneficial in that they are capable of separating cells into very small populations, however they have no means of sorting different types of cells into specific wells. And since the wells “trap” the cells within physical barriers, growth and motility of the cells are restricted.
A more improved version of this technology uses micropatterned “holes” in Polydimethylsiloxane (PDMS) or another elastomeric polymer, which is laid on top of a growth substrate before cell seeding. The cells are deposited onto the array of holes such that they land and attach on the substrate, then the PDMS hole array is removed, leaving the cells patterned only in areas where the holes extended to the substrate (see, Emanuele Ostuni, Ravi Kane, Christopher S. Chen, Donald E. Ingber, and, and George M. Whitesides, Patterning Mammalian Cells Using Elastomeric Membranes, Langmuir 16 (20), 2000, 7811-7819). This allows the cells to grow freely on an unconfined surface, however it does not provide a means for seeding multiple cell types simultaneously. Other groups have used microcontact printing and other methods to pattern cell-adhesive chemicals and proteins onto substrates, causing cells to only attach to those regions containing the chemical (see Ravi S Kane, Shuichi Takayama, Emanuele Ostuni, Donald E Ingber, George M Whitesides, Patterning Proteins and Cells Using Soft Lithography, Biomaterials, 20 (23-24), 1999, 2363-2376). Though this allows cells to grow freely on a flat surface, the surface has been modified chemically in specific areas, causing potential variation in how the cell population grows, migrates, and proliferates. It is possible to have different cell types attach to certain areas by patterning chemicals that interact preferentially with certain cells. However, many of the chemicals or proteins used in this approach interact favorably with many types of cells. This is especially true if the cell populations used are from the same organ (e.g., the brain or spinal cord).
This invention addresses all of the aforementioned shortcomings in a relatively simple and easy to use design. The proposed funnel design allows for multiple cell types to easily be seeded into macroscale openings at the top of the device, then the openings narrow substantially such that the cells land on the surface separated by distances as small as several microns. Once the cells attach, the funnel is removed, producing a flat, unmodified surface with cells of one or more types localized to specific regions of the substrate.